Wednesday, June 10, 2015

256 - Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization

So we saw that Azotobacter vinelandii seems to have multiple copies of its chromosome in each cell (253,255), but we also seemed to see that it doesn't exhibit the behavior expected of a polyploid organism, at least sometimes (445). Other times it might. One proposed explanation is that it has some mechanism by which it homogenizes its genotype, a type of homologous recombination or "homogenotization."

A possible mechanism for this is the RecF system found in E. coli, which is involved in repairing the genome after recombination. A. vinelandii has a homologous system, so this study investigated the phenotype of cells with it knocked out.

What They Saw
They knocked out recF, recA, or both from A. vinelandii UW (aka CA), by inserting tetracycline resistance cassettes. This was confirmed by Southern blotting. Actually they already had a recA knockout, so they just made that a double-knockout. That was somewhat tricky, since the recombination frequency is much lower than in the wild-type; that's not very surprising, since RecA is probably pretty important for recombination.

They found that knocking out recF seemed to impair recombination too (also not surprising), though not as much as lack of recA. With both gone, the recombination proficiency was even lower. They saw similar results with UV sensitivity (and thus ability to repair DNA damage).

Finally, the question of homogenotization: to test this, they knocked out the nifLA genes (which regulate/activate nitrogenase) by inserting a kanamycin resistance cassette, expecting that if RecA or RecF were involved in homogenotization, the lack of them would mean that it would be easy to isolate cells with kan resistance in some chromosome copies and yet capable of nitrogen fixation (because wild-type nifLA is still present in other copies to turn on the nitrogenase). This approach, again, seems questionable to me due to the presence of multiple nitrogenases. I'm not sure if NifLA are necessary to regulate all three versions, or just the Mo one.

However, they didn't find any transformants from any of the strains (wild-type or recA or recF or double-knockouts) that were both kan-resistant and nitrogen-fixing. So they conclude that these deleted genes aren't involved in so-called homogenotization, since their absence didn't make a difference. Is it possible that the homogenotization happened when cells divided and only those with kan resistance survived? I'm not sure that question was addressed. It doesn't seem known if progeny get just one copy and then make more, or if copies are equally partitioned. I suppose the latter makes more sense, since it is binary fission, as far as we know.

Also, they did say that antibiotics weren't necessary to prevent nitrogen fixation phenotypes, so I guess that addresses my question. And they did more Southern blotting to show that there was a cassette inserted in the nifLA locus in all the transformants, with no wild-type alleles visible.

I'm not convinced that homogenotization is a real thing, but I'm not yet sure how to explain these data. Somehow it seems like A. vinelandii has many copies of its genome, but it's just as possible to transform all of them the same way as to transform just some. Guess I'll keep reading.

Badran, H., Sohoni, R., Venkatesh, T. V. & Das, H. K. Construction of a recF deletion mutant of Azotobacter vinelandii and its characterization. FEMS Microbiology Letters 174, 363–369 (1999).

No comments:

Post a Comment